The vertebrate eye lens consists of long, hexagonally-shaped units, termed lens fiber cells, which are derived from an epithelial monolayer by a process of cellular elongation (lens differentiation). Attention has focused recently on the role of the cytoskeleton in age-related changes in the morphology and function of these fiber cells. However, little is known about the molecular mechanism involved or how this interaction modulates lens cell differentiation and function. Preliminary results have shown that differentiating lens fiber cells in situ express polypeptides related to erythrocyte spectrin which, by analogy to the function of spectrin in the erythrocyte, may be suitable candidates for mediating linkage of actin filaments to the plasma membrane and in the maintenance of cell shape. Experiments outlined in this grant proposal are aimed at elucidating the function of spectrin in lens differentiation and aging. Since differentiating lens fiber cells are laid down in layers at the periphery of the lens and are then displaced toward the center, a gradient of cells of increasing age is formed in which distinct layers can be recognized. These layers can be separated by microdissection and used to determine changes in the synthesis, distribution and physicochemical properties of spectrin that occur during lens differentiation and aging. The synthesis of spectrin during lens differentiation will be determined by the incorporation of 35S-methionine into newly synthesized protein in situ and in vitro. The distribution of spectrin will be examined using immunocytochemical techniques involving indirect immunofluorescence microscopy and electron microscopy on plasma membranes. Changes in the physicochemical properties of spectrin and its association with the plasma membrane and actin filaments during lens differentiation will be followed also by extraction of plasma membranes under a variety of ionic, detergent and denaturing conditions. Together these results will provide, for the first time, an insight into the molecular mechanism involved in the interaction between the cytoskeleton and the lens cell plasma membrane, and relate changes in the distribution and properties of lens spectrin to cellular elongation and increased rigidity of the plasma membrane during lens differentiation.